Sustainable production of a polysaccharide-based glycoprotein by simultaneous conversion of glucose and glycerol in engineered Escherichia coli

Abstract

Urinary tract infection (UTI) is a major infectious disease commonly induced by uropathogenic Escherichia coli (UPEC), of which a prevalent serogroup is the mostly antibiotic-resistant O4. Therefore, protective vaccines against UPEC infection, especially serogroup O4, are needed. Polysaccharide-based glycoproteins are promising candidates for development of anti-bacterial vaccines; however, current chemical and chemoenzymatic syntheses are costly and environmentally unfriendly, because of the complex ligation strategies and chemical reagents required. Development of efficient microbial cell factories, using cheap, renewable feedstocks and avoiding the use of toxic reagents would reduce toxic waste generation and environmental concerns. An O-glycosylation Escherichia coli chassis was developed to install the O-antigen gene cluster to biosynthesize a polysaccharide-based glycoprotein, by co-utilization of a glucose/glycerol mixture with high synergistic metabolic efficiency. Towards this goal, 32 genes related to competing pathways were deleted from E. coli MG1655 and 28 genes relevant to the biosynthesis of the glycoprotein were introduced, resulting in the successful production of the target recombinant glycoprotein. Subsequently, the availability of the intracellular sugar derivative precursors dTDP-L-Rha and UDP-L-FucNAc was optimized by tuning the expression of the corresponding enzymes. Further optimization of fermentation conditions resulted in a glycoprotein titre of 27.71 mg L⁻¹. MS and EMR confirmed the precise biosynthesis of the target glycoprotein and highly efficient glycosylation. Finally, vaccination with the glycoprotein stimulated a strong immune response in mice, which markedly reduced the bacterial burden in the bladder and kidneys after UPEC challenge. This study provides guidance for the development of a sustainable and environmentally friendly manufacturing platform for specific high-value therapeutic glycoproteins.